1. Field of the Invention
The present invention relates to fluorescein derivatives which are useful for reagents for measurement of nitrogen monoxide. It also relates to reagents for measuring nitrogen monoxide which comprise said compounds.
2. Related Art
Nitrogen monoxide (NO) is an unstable radical having a short life, and it has been elucidated that nitrogen monoxide has important functions as a physiologically active substance in a living body (featured in Gendai Kagaku (Chemistry Today), April, 1994). Methods for measuring nitrogen monoxide are mainly classified into (a) indirect methods where oxidative degradation products of nitrogen monoxide such as NO.sub.2 -- or NO.sub.3 -- are measured, and (b) methods where nitrogen monoxide is directly measured. The direct methods have been focused from the standpoint that they achieve detection and quantification of nitrogen monoxide under physiological condition. However, no measuring method has been developed to date that has sufficient specificity and high sensitivity and is applicable to an in vitro system.
For example, a chemiluminescence method which utilizes luminescence emitted during an ozonic oxidation of NO radicals (Palmer, R. M., et al., Nature, 327, pp.524-526, 1987); a method which comprises the step of measuring an absorption spectrum of metHb that is produced by an oxidation of oxyhemoglobin (O.sub.2 Hb) (Kelm, M., et al., Circ. Res.66, pp.1561-1575, 1990); a method which comprises the step of measuring electric current generated during an oxidation by means of electrodes that are inserted into a tissue (Shibuki, K., Neurosci. Res.9, pp.69-76, 1990; Malinski, T., Nature, 356, pp.676-678, 1992); and the Griess reaction method (Green, L. C., et al., Anal. Biochem., 126, pp.131-138, 1992) are known as typical methods (as reviews, see, "3. Method for the measurement of NO," by Tetsuo Nagano, pp.42-52, "Approach from the Latest Medicine 12, NO" edited by Noboru Toda, published by Medical View Co., Ltd.; and Archer, S., FASEB J., 7, pp.349-360, 1993).
The Griess reaction method comprises a detection step that utilizes the azo coupling between naphthylethylenediamine and a diazonium salt compound formed with NO.sub.2 -- which is generated by the oxidation of nitrogen monoxide radicals. This method is advantageous because it does not require particular apparatuses or techniques, although nitrogen monoxide radicals are not directly measured in this method. In addition, NO.sub.3 -- can also be measured after being reduced to NO.sub.2 -- by using cadmium (Stainton, M. P., Anal. Chem., 46, p.1616, 1974; Green, L. C., et al., Anal. Biochem., 126, pp.131-138, 1982) or hydrazine (Sawicki, C. R. and Scaringelli, F. P., Microchem. J., 16, pp.657-672, 1971), and accordingly, the method also has characteristic feature that it enables the measurement of metabolites related to nitrogen monoxide.
2,3-Diaminonaphthalene has also been known as a reagent for measuring nitrogen monoxide by detecting NO.sub.2 --, as in a similar manner to Griess reaction method. This reagent reacts with NO.sub.2 -- under an acidic condition to form a fluorescent adduct, i.e., naphthalenetriazole (chemical name: 1-H!-naphtho2,3-d!triazole) (Wiersma, J. H., Anal. Lett., 3, pp.123-132, 1970). The reaction conditions of 2,3-diaminonaphthalene and NO.sub.2 -- have been detailedly studied, and it has been found that the reaction proceeds most rapidly at a pH not higher than 2, and completes within about 5 minutes at room temperature (Wiersma, J. H., Anal. Lett., 3, pp.123-132, 1970; Sawicki, C. R., Anal. Lett., 4, pp.761-775, 1971). The resulting adduct emits fluorescence most efficiently at a pH not lower than 10 (Damiani, P. and Burini, G., Talanta, 8, pp.649-652, 1986).
The method for measuring nitrogen monoxide by using the above 2,3-diaminonaphthalene has characteristic features of 50- to 100-fold higher sensitivity compared to the Griess reaction method and of detection limit at approximately several tens nM (Misko, T. P., Anal. Biochem. 214, pp.11-16, 1993). This method is highly advantageous because it does not need particular apparatuses or techniques and can be conveniently carried out (as a review of the aforementioned method, see, DOJIN News. No. 74, Information, "A reagent for the determination of NO: 2,3-diaminonaphthalene," Dojindo Laboratories Inc., 1995). However, this method does not utilize nitrogen monoxide, per se, but it utilizes an oxidation product, i.e., NO.sub.2 --, as a reactant. Accordingly, the method is considered as an indirect method when compared to those including direct measurement of nitrogen monoxide. Furthermore, because the reaction of 2,3-diaminonaphthalene with NO.sub.2 -- is progressed under a strongly acidic condition (pH not higher than 2), the method has a problem in that it cannot be employed for detection or quantification of nitrogen monoxide under a physiological condition.
The inventors of the present invention conducted researches to provide a means that enables direct and highly sensitive measurement of nitrogen monoxide under a physiological condition, and as a result, they found that nitrogen monoxide can efficiently react with 2,3-diaminonaphthalene or its derivatives, even under a neutral condition, in the presence of an oxygen source such as dissolved oxygen or oxide compounds (e.g., PTIO and derivatives thereof such as carboxy-PTIO), and a fluorescent naphthalenetriazole or a derivative thereof is obtained. They also found that a method for measuring nitrogen monoxide utilizing the above reaction has extremely high detection sensitivity, and can achieve accurate quantification of very small amount of nitrogen monoxide (see, the specification of Japanese Patent Application No. Hei 7-189978).
However, the aforementioned method utilizing 2,3-diaminonaphthalene requires the irradiation with excitation light having a short wavelength of approximately 370-390 nm for the detection of fluorescence, and this may cause damages to cells and/or tissues in a measurement system. Strong autofluorescence of cells, per se, may also possibly affect this measurement, and moreover, there is a problem that a fluorescence filter provided on a usual fluorescence microscope fails to sufficiently cut off excitation light during fluorescence measurement. In addition, the fluorescent triazole compound formed from the 2,3-diaminonaphthalene has rather insufficient fluorescence intensity, and therefore, it is difficult to achieve accurate measurement of intracellular fluorescence of an individual cell by ordinary fluorescence microscopy. Since 2,3-diaminonaphthalene itself has a simple chemical structure, there is also a problem that the compound is not suitable as a fundamental structure for various chemical modifications to achieve intracellular localization of a reagent.